Cholesterol, triacylglycerol and other lipids are transported in body fluids by lipoproteins which are classified according to their density: chylomicrons, chylomicron remnants, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). A lipoprotein is a particle with a nucleus made of hydrophobic lipids which nucleus is surrounded by a cover of polar lipids and apoproteins. Lipoproteins have two functions: they solubilize highly hydrophobic lipids and they comprise signals which help regulate the transport of specific lipids to specific cells and tissues.
Triacylglycerol, cholesterol and other lipids taken up with food are transported with chylomicrons from the intestine to fatty tissue and to the liver.
Endogenous triacylglycerols are synthesized in the liver and transported by VLDL. Lipase helps release triacylglycerols from the VLDL; the substance remaining after such release is high in cholesterol and called the IDL. The IDL may be taken up and metabolized by the liver or transformed into LDL, which is the main cholesterol carrier of the blood. The function of these LDL particles is to transport cholesterol to peripheral tissues and to help control de novo cholesterol production. It has been shown that high levels of LDL-Cholesterol in the blood correlate with atherosclerosis which is a progressive disease characterized in part by sedimentation of lipids in inner walls of arteries, particularly of coronary arteries. It has also been shown that a high blood level of LDL-Cholesterol correlates with coronary heart disease. Also, a negative correlation exists between blood levels of HDL cholesterol and coronary heart disease.
The level of total cholesterol in blood, which is the sum of HDL-Cholesterol, LDL-Cholesterol, VLDL-Cholesterol and chylomicron-Cholesterol, is not considered an adequate indicator of the risk of coronary heart disease and atherosclerosis. It is important to determine the amount of LDL-Cholesterol in a patient, for instance as an indication of such risk or for other reasons that may be related to health. Various methods of measuring LDL-cholesterol have been described in the state of the art:
1. Electrophoretic separation with following visualization of lipoprotein bands by polyanion precipitation and conversion of turbidity units into cholesterol values.
2. Ultracentrifugation
This method requires special apparatus and is very time consuming.
3. LDL-Cholesterol can be furthermore calculated with the help of the Friedewald formula whereby the total cholesterol, HDL cholesterol and triglycerol values are determined in a sample. LDL-Cholesterol=total cholesterol−HDL cholesterol−⅕ triglyceride. However, this calculation contains an error of about 10%.
4. Precipitation reactions.
Various reactions are described, for example a method where LDL is precipitated with the help of lectin and the amount of cholesterol before and after precipitation is determined and the amount of LDL-Cholesterol is calculated.
Handbook of Lipoprotein Testing, 2d ed., Eds. Nader Rifai, G. Russell Warnick, Marek H. Dominiczak: AACC Press (2000), Chapters 9 and 12.
However, these methods are time consuming and not suitable for routine and high throughput testing, or for testing by a subject for instance at home, without a need for professional assistance. Therefore, there is a need for more efficient LDL-Cholesterol tests. Various publications relate to these aspects:
U.S. Pat. Nos. 5,532,172 and 4,746,605 both relate to a method for determining the LDL-Cholesterol concentration in a sample, whereby in a first step HDL is separated with the help of HDL specific antibodies. After separation, the remaining LDL-Cholesterol is determined.
U.S. Pat. No. 6,057,118 discloses a method for measuring the LDL-Cholesterol concentration in a sample whereby a surfactant is added to the sample to block LDL-cholesterol. Other forms of cholesterol such as HDL-Cholesterol and VLDL-Cholesterol are then reacted with a cholesterol-assaying enzyme reagent, and the remaining amount of LDL-cholesterol is determined thereafter. By measuring cholesterol at a certain period of time after addition of the surfactant, only the LDL-Cholesterol is determined.
U.S. Pat. No. 5,807,696 discloses a method wherein LDL is inhibited, HDL-Cholesterol is used by reaction with enzymes, after which the remaining LDL-Cholesterol is determined.
U.S. Pat. No. 5,888,827 discloses a test system in liquid phase where the addition of a sugar compound and a protein solubilizing agent results in different reactivities of the different compounds HDL, LDL, VLDL and chylomicrons. After selecting and adding a combination of two reagents a chromogenic substance is added with which the LDL-Cholesterol is determined.
U.S. Pat. No. 4,544,630 discloses a method in which LDL-Cholesterol is determined at a specific time. By adding a specific surfactant at a pH of 6.5 to 8.0, LDL-Cholesterol reacts before HDL-Cholesterol. Therefore, by measuring the amount of cholesterol at a defined moment, only the amount of LDL-Cholesterol is determined. This method requires a meter and computer for analysis of the amount of LDL-cholesterol in a sample.
U.S. Pat. No. 5,804,450 discloses a liquid method and reagent for determining LDL in serum samples by adding an LDL precipitating agent, an LDL aggregating agent and a zwitterionic detergent to a sample, and then detecting LDL aggregates.
U.S. Pat. No. 5,795,786 discloses a liquid method and reagent for determining LDL in serum samples by adding an LDL aggregating agent and taking turbidimetric measurements of the LDL aggregate.
A similar method is described in U.S. Pat. No. 6,524,864 where a test strip is provided with an LDL precipitating agent, such as dextran, on a first layer after which HDL passes through a second layer and reacts on a reaction pad containing cholesterol esterase, cholesterol oxidase, peroxidase, surfactants and TMB (tetramethylbenzidine chromogen). A difference between total cholesterol and HDL cholesterol values yields LDL-Cholesterol values.
U.S. Pat. No. 5,340,539 discloses a method for using a “thermometer”-shaped device to determine the amount of total cholesterol or total cholesterol ester in a sample.
U.S. 2004/0126830 A1 discloses indirectly measuring LDL-cholesterol by measuring total cholesterol and the sum total of HDL, VLDL and chylomicron cholesterol, and then calculating the amount of LDL-cholesterol by subtracting from the measured amount of total cholesterol the measured amount of the sum total of HDL, VLDL and chylomicron cholesterol. The sum total of cholesterol of HDL, VLDL and chylomicrons is measured by adding a surfactant to inhibit LDL-cholesterol and allow for the detection of HDL, VLDL and chylomicron cholesterol before LDL-cholesterol is detected.
U.S. 2004/0023400 discloses an assay for measuring HDL cholesterol by using a surfactant to increase the solubility of HDL and another surfactant to inhibit lipoprotein other than HDL from dissolving.